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Animal flesh foods are rich in bioavailable iron but infrequently consumed by young children. We aimed to determine whether flesh food intake was associated with iron and anaemia status among 585 Malawian infants enroled in a 6-month egg-feeding trial. The percentage of days of small fish, large fish and meat consumption were assessed through weekly 7-day animal-source food screeners. Grams of intake were assessed through 24-h recalls conducted at 6-9, 9-12 and 12-15 months of age. Plasma ferritin, soluble transferrin receptor (sTfR) and haemoglobin concentrations were measured at 6-9 and 12-15 months of age. Iron biomarkers were adjusted for inflammation during analysis. At enrolment, each flesh food category was consumed by

BACKGROUND: Murine data suggest that the placenta downregulates ferroportin (FPN) when iron is limited to prioritize iron for its own needs. Human data on the impact of maternal and neonatal iron status on placental FPN expression are conflicting. OBJECTIVES: This study aimed to identify determinants of placental FPN protein abundance and to assess the utility of the placental iron deficiency index (PIDI) as a measure of maternal/fetal iron status in newborns at high risk for anemia. METHODS: Placental FPN protein abundance was measured by western blots in placentae collected from 133 neonates born to adolescents (17.4 ± 1.1 y) carrying singletons (delivery gestational age [GA]: 39.9 ± 1.3 wk) and from 130 neonates born to 65 females (30.4 ± 5.2 y) carrying multiples (delivery GA: 35.0 ± 2.8 wk). Placental FPN and the PIDI (FPN:transferrin receptor 1) were evaluated in relation to neonatal and maternal iron-related markers (hemoglobin [Hb], serum ferritin [SF], soluble transferrin receptor [sTfR], total body iron [TBI], hepcidin, erythropoietin [EPO], erythroferrone). RESULTS: FPN protein was detected in all placentae delivered between 25 and 42 wk GA. Placental FPN protein abundance was associated with neonatal iron and erythropoietic markers (EPO: β: 0.10; 95% confidence interval [CI]: 0.06, 0.35; sTfR: β: 0.20; 95% CI: 0.03, 0.18; hepcidin: β: -0.06; 95% CI: -0.13, -0.0003; all P < 0.05). Maternal sTfR was only indirectly associated with placental FPN, with neonatal sTfR as the mediator (β-indirect: 0.06; 95% CI; 0.03, 0.11; P = 0.003). The PIDI was associated with neonatal Hb (β: -0.02; 95% CI: -0.03, -0.003), EPO (β: 0.07; 95% CI: 0.01, 0.14), and sTfR (β: 0.13; 95% CI: 0.004, 0.3) and with maternal SF (β: 0.08, 95% CI: 0.02, 0.14), TBI (β: 0.02; 95% CI: 0.009, 0.04), EPO (β: -0.10; 95% CI: -0.19, -0.01), sTfR (β: -0.16: 95% CI: -0.27, -0.06), and hepcidin (β: 0.05; 95% CI: 0.002, 0.11) at delivery (all P < 0.05). CONCLUSIONS: Placental FPN abundance was positively associated with neonatal indicators of increased erythropoietic activity and poor iron status. The PIDI was associated with maternal and neonatal iron-related markers but in opposite directions. More data are needed from a lower-risk normative group of females to assess the generalizability of findings. These trials were registered at clinicaltrials.gov as NCT01019902 and NCT01582802.

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with the development of hematopoietic cancers of B-lymphocyte origin, including AIDS-related non-Hodgkin lymphoma (AIDS-NHL). Primary infection of B-cells with EBV results in their polyclonal activation and immortalization. The transferrin receptor 1 (TfR1), also known as CD71, is important for iron uptake and regulation of cellular proliferation. TfR1 is highly expressed in proliferating cells, including activated lymphocytes and malignant cells. We developed a mouse/human chimeric antibody targeting TfR1 (ch128.1/IgG1) that has previously shown significant antitumor activity in immunosuppressed mouse models bearing human malignant B-cells, including multiple myeloma and AIDS-NHL cells. In this article, we examined the effect of targeting TfR1 to inhibit EBV-driven activation and growth of human B-cells in vivo using an immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ [NOD/SCID gamma (NSG)] mouse model. Mice were implanted with T-cell-depleted, human peripheral blood mononuclear cells (PBMCs), either without EBV (EBV-), or exposed to EBV in vitro (EBV+), intravenously via the tail vein. Mice implanted with EBV+ cells and treated with an IgG1 control antibody (400 μg/mouse) developed lymphoma-like growths of human B-cell origin that were EBV+, whereas mice implanted with EBV+ cells and treated with ch128.1/IgG1 (400 μg/mouse) showed increased survival and significantly reduced inflammation and B-cell activation. These results indicate that ch128.1/IgG1 is effective at preventing the growth of EBV+ human B-cell tumors in vivo, thus, indicating that there is significant potential for agents targeting TfR1 as therapeutic strategies to prevent the development of EBV-associated B-cell malignancies. SIGNIFICANCE: An anti-TfR1 antibody, ch128.1/IgG1, effectively inhibits the activation, growth, and immortalization of EBV+ human B-cells in vivo, as well as the development of these cells into lymphoma-like tumors in immunodeficient mice.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor κB (NF-κB) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

Iron deficiency is common worldwide and is associated with adverse pregnancy outcomes. The increasing prevalence of indiscriminate iron supplementation during pregnancy also raises concerns about the potential adverse effects of iron excess. We examined how maternal iron status affects the delivery of iron to the placenta and fetus. Using mouse models, we documented maternal homeostatic mechanisms that protect the placenta and fetus from maternal iron excess. We determined that under physiological conditions or in iron deficiency, fetal and placental hepcidin did not regulate fetal iron endowment. With maternal iron deficiency, critical transporters mediating placental iron uptake (transferrin receptor 1 [TFR1]) and export (ferroportin [FPN]) were strongly regulated. In mice, not only was TFR1 increased, but FPN was surprisingly decreased to preserve placental iron in the face of fetal iron deficiency. In human placentas from pregnancies with mild iron deficiency, TFR1 was increased, but there was no change in FPN. However, induction of more severe iron deficiency in human trophoblast in vitro resulted in the regulation of both TFR1 and FPN, similar to what was observed in the mouse model. This placental adaptation that prioritizes placental iron is mediated by iron regulatory protein 1 (IRP1) and is important for the maintenance of mitochondrial respiration, thus ultimately protecting the fetus from the potentially dire consequences of generalized placental dysfunction.

Dinoflagelates and cyanobacteria produce saxitoxin (STX), a lethal bis-guanidinium neurotoxin causing paralytic shellfish poisoning. A number of metazoans have soluble STX-binding proteins that may prevent STX intoxication. However, their STX molecular recognition mechanisms remain unknown. Here, we present structures of saxiphilin (Sxph), a bullfrog high-affinity STX-binding protein, alone and bound to STX. The structures reveal a novel high-affinity STX-binding site built from a "proto-pocket" on a transferrin scaffold that also bears thyroglobulin domain protease inhibitor repeats. Comparison of Sxph and voltage-gated sodium channel STX-binding sites reveals a convergent toxin recognition strategy comprising a largely rigid binding site where acidic side chains and a cation-π interaction engage STX. These studies reveal molecular rules for STX recognition, outline how a toxin-binding site can be built on a naïve scaffold, and open a path to developing protein sensors for environmental STX monitoring and new biologics for STX intoxication mitigation.

Zika virus (ZIKV) infection is implicated in severe fetal developmental disorders, including microcephaly. MicroRNAs (miRNAs) post-transcriptionally regulate numerous processes associated with viral infection and neurodegeneration, but their contribution to ZIKV pathogenesis is unclear. We analyzed the mRNA and miRNA transcriptomes of human neuronal stem cells (hNSCs) during infection with ZIKV MR766 and Paraiba strains. Integration of the miRNA and mRNA expression data into regulatory interaction networks showed that ZIKV infection resulted in miRNA-mediated repression of genes regulating the cell cycle, stem cell maintenance, and neurogenesis. Bioinformatics analysis of Argonaute-bound RNAs in ZIKV-infected hNSCs identified a number of miRNAs with predicted involvement in microcephaly, including miR-124-3p, which dysregulates NSC maintenance through repression of the transferrin receptor (TFRC). Consistent with this, ZIKV infection upregulated miR-124-3p and downregulated TFRC mRNA in ZIKV-infected hNSCs and mouse brain tissue. These data provide insights into the roles of miRNAs in ZIKV pathogenesis, particularly the microcephaly phenotype.

We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation and time-gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely-available software, has the advantage of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging and could be extended to applications as diverse as image-guided surgery or optical tomography.

PurposePancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, with a 5-year survival rate of less than 10%. Physicians often rely on biopsy or CT to guide treatment decisions, but these techniques fail to reliably measure the actions of therapeutic agents in PDAC. KRAS mutations are present in >90% of PDAC and are connected to many signaling pathways through its oncogenic cascade, including extracellular regulated kinase (ERK) and MYC. A key downstream event of MYC is transferrin receptor (TfR), which has been identified as a biomarker for cancer therapeutics and imaging.Experimental designIn this study, we aimed to test whether zirconium-89 transferrin ([89Zr]Zr-Tf) could measure changes in MYC depending on KRAS status of PDAC, and assess target engagement of anti-MYC and anti-ERK-targeted therapies.ResultsMice bearing iKras*p53* tumors showed significantly higher (P < 0.05) uptake of [89Zr]Zr-Tf in mice withdrawn from inducible oncogenic KRAS. A therapy study with JQ1 showed a statistically significant decrease (P < 0.05) of [89Zr]Zr-Tf uptake in drug versus vehicle-treated mice bearing Capan-2 and Suit-2 xenografts. IHC analysis of resected PDAC tumors reflects the data observed via PET imaging and radiotracer biodistribution.ConclusionsOur study demonstrates that [89Zr]Zr-Tf is a valuable tool to noninvasively assess oncogene status and target engagement of small-molecule inhibitors downstream of oncogenic KRAS, allowing a quantitative assessment of drug delivery.

Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer's disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Five and a half month old male APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with saline ( n = 10) or the BBB-penetrable EPO ( n = 10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline ( n = 8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aβ peptide load, synaptic loss, BBB disruption, microglial activation, and microhemorrhages. APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aβ peptide number ( p < 0.05) and immune-positive area ( p < 0.05), a decrease in hippocampal synaptic loss ( p < 0.05) and cortical microglial activation ( p < 0.001), and improved spatial memory ( p < 0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aβ load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.

Noninvasive tools that target tumor cells could improve the management of glioma. Cancer generally has a high demand for Fe(III), an essential nutrient for a variety of biochemical processes. We tested whether 68Ga-citrate, an Fe(III) biomimetic that binds to apo-transferrin in blood, detects glioma in preclinical models and patients using hybrid PET/MRI. Mouse PET/CT studies showed that 68Ga-citrate accumulates in subcutaneous U87MG xenografts in a transferrin receptor-dependent fashion within 4 hours after injection. Seventeen patients with WHO grade III or IV glioma received 3.7-10.2 mCi 68Ga-citrate and were imaged with PET/MR 123-307 minutes after injection to establish that the radiotracer can localize to human tumors. Multiple contrast-enhancing lesions were PET avid, and tumor to adjacent normal white matter ratios were consistently greater than 10:1. Several contrast-enhancing lesions were not PET avid. One minimally enhancing lesion and another tumor with significantly reduced enhancement following bevacizumab therapy were PET avid. Advanced MR imaging analysis of one patient with contrast-enhancing glioblastoma showed that metabolic hallmarks of viable tumor spatially overlaid with 68Ga-citrate accumulation. These early data underscore that high-grade glioma may be detectable with a radiotracer that targets Fe(III) transport.

The tumor-promoting functions of neutrophils have been mainly attributed to induction of tumor angiogenesis or suppression of anticancer immunity. However, a direct impact of neutrophils on tumor cell growth and metastasis remains largely uncharacterized. Here, we coupled a proteomic approach with a functional screen to interrogate the secretome of tumor-associated neutrophils. Surprisingly, the iron-transporting protein transferrin was identified as the major mitogen for tumor cells secreted by neutrophils. Depletion of neutrophils inhibited lung metastasis and transferrin production in the metastatic microenvironment. Deletion of transferrin receptor suppressed growth of lung-colonizing tumor cells. Also, media conditioned by neutrophils isolated from metastatic breast cancer patients stimulated growth of human breast cancer cells, an effect that was largely abolished by transferrin immunodepletion. We identified GM-CSF, which is produced primarily by tumor cells, as a selective inducer of de novo transferrin synthesis in neutrophils through the Jak/Stat5β pathway. GM-CSF neutralization or inhibition of Jak kinases curtailed neutrophil transferrin expression in vitro and in vivo as well as cancer metastasis. Thus, transferrin provides a mechanistic link between neutrophils and metastatic growth owing to the ability of tumor-infiltrating neutrophils to locally deliver this growth-promoting protein in response to GM-CSF stimulation. Our study identifies neutrophil-derived transferrin as a key regulator of metastatic tumor cell growth and a therapeutic target for antimetastatic treatment.

Iron is an essential micronutrient for most microbes and their hosts. Mammalian hosts respond to infection by inducing the iron-regulatory hormone hepcidin, which causes iron sequestration and a rapid decrease in the plasma and extracellular iron concentration (hypoferremia). Previous studies showed that hepcidin regulation of iron is essential for protection from infection-associated mortality with the siderophilic pathogens Yersinia enterocolitica and Vibrio vulnificus However, the evolutionary conservation of the hypoferremic response to infection suggests that not only rare siderophilic bacteria but also common pathogens may be targeted by this mechanism. We tested 10 clinical isolates of Escherichia coli from children with sepsis and found that both genetic iron overload (by hepcidin-1 knockout [HKO]) and iatrogenic iron overload (by intravenous iron) potentiated infection with 8 out of the 10 studied isolates: after peritoneal injection of E. coli, iron-loaded mice developed sepsis with 60% to 100% mortality within 24 h, while control wild-type mice suffered 0% mortality. Using one strain for more detailed study, we show that iron overload allows rapid bacterial multiplication and dissemination. We further found that the presence of non-transferrin-bound iron (NTBI) in the circulation is more important than total plasma or tissue iron in rendering mice susceptible to infection and mortality. Postinfection treatment of HKO mice with just two doses of the hepcidin agonist PR73 abolished NTBI and completely prevented sepsis-associated mortality. We demonstrate that the siderophilic phenotype extends to clinically common pathogens. The use of hepcidin agonists promises to be an effective early intervention in patients with infections and dysregulated iron metabolism.

The transferrin receptor 1 (TfR1) is an attractive target for Ab-mediated cancer therapy. We previously developed a mouse/human chimeric IgG3 Ab (ch128.1) targeting human TfR1, which exhibits direct in vitro cytotoxicity against certain human malignant B cells through TfR1 degradation and iron deprivation. ch128.1 also demonstrates exceptional antitumor activity against the B cell malignancy multiple myeloma (MM) in xenograft models of SCID-Beige mice bearing either disseminated ARH-77 or KMS-11 cells in an early disease setting. Interestingly, this activity is observed even against KMS-11 cells, which show no sensitivity to the direct cytotoxic activity of ch128.1 in vitro. To understand the contributions of the Fc fragment, we generated a ch128.1 mutant with impaired binding to FcγRs and to the complement component C1q, which retains binding to the neonatal Fc receptor. We now report that this mutant Ab does not show antitumor activity in these two MM models, indicating a crucial role of the Fc fragment in the antitumor activity of ch128.1, which can be attributed to effector functions (Ab-dependent cell-mediated cytotoxicity, Ab-dependent cell-mediated phagocytosis, and/or complement-dependent cytotoxicity). Interestingly, in the KMS-11 model, complement depletion does not affect protection, whereas macrophage depletion does. Consistent with this observation, we found that ch128.1 induces Ab-dependent cell-mediated cytotoxicity and Ab-dependent cell-mediated phagocytosis against KMS-11 cells in the presence of murine bone marrow-derived macrophages. Finally, we found that ch128.1 therapy effectively increases survival in a late MM disease setting. Our results suggest that macrophages play a major role in ch128.1-mediated antitumor protection in our models and that ch128.1 can be effective against human B cell malignancies such as MM.

Background & aimsMulticopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/- ) or intestine alone (Hephint/int ) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.MethodsMice lacking both HEPH and CP (Heph-/-Cp-/- ) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/- ) were generated and phenotyped.ResultsHeph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.ConclusionsThese studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

The current standard for breast PET imaging is 18F-FDG. The heterogeneity of 18F-FDG uptake in breast cancer limits its utility, varying greatly among receptor status, histopathologic subtypes, and proliferation markers. 18F-FDG PET often exhibits nonspecific internalization and low specificity and sensitivity, especially with tumors smaller than 1 cm3 MYC is a protein involved in oncogenesis and is overexpressed in triple-negative breast cancer (TNBC). Increased surface expression of transferrin receptor (TfR) is a downstream event of MYC upregulation and has been validated as a clinically relevant target for molecular imaging. Transferrin labeled with 89Zr has successfully identified MYC status in many cancer subtypes preclinically and been shown to predict response and changes in oncogene status via treatment with small-molecule inhibitors that target MYC and PI3K signaling pathways. We hypothesized that 89Zr-transferrin PET will noninvasively detect MYC and TfR and improve upon the current standard of 18F-FDG PET for MYC-overexpressing TNBC. Methods: In this study, 89Zr-transferrin and 18F-FDG imaging were compared in preclinical models of TNBC. TNBC cells (MDA-MB-157, MDA-MB-231, and Hs578T) were treated with bromodomain-containing protein 4 (BRD4) inhibitors JQ1 and OTX015 (0.5-1 μM). Cell proliferation, gene expression, and protein expression were assayed to explore the effects of these inhibitors on MYC and TfR. Results: Head-to-head comparison showed that 89Zr-transferrin targets TNBC tumors significantly better (P < 0.05-0.001) than 18F-FDG through PET imaging and biodistribution studies in MDA-MB-231 and MDA-MB-157 xenografts and a patient-derived xenograft model of TNBC. c-Myc and TfR gene expression was decreased upon treatment with BRD4 inhibitors and c-MYC small interfering RNA (P < 0.01-0.001 for responding cell lines), compared with vehicle treatment. MYC and TfR protein expression, along with receptor-mediated internalization of transferrin, was also significantly decreased upon drug treatment in MDA-MB-231 and MDA-MB-157 cells (P < 0.01-0.001). Conclusion:89Zr-transferrin targets human TNBC primary tumors significantly better than 18F-FDG, as shown through PET imaging and biodistribution studies. 89Zr-transferrin is a useful tool to interrogate MYC via TfR-targeted PET imaging in TNBC.

Background: Proteinuria is an unfavorable clinical condition highly associated with a risk of renal and cardiovascular disease in chronic kidney disease (CKD). However, whether all proteinuria forms are linked to renal impairment are still unclear. Cubilin is an endocytic receptor highly expressed in renal proximal tubules mediating uptake of albumin, transferrin and α1-microglobulin.Methods: Exome sequencing method initially identified candidate genes. With the application of exome sequencing combined with Sanger sequencing, we further focused on CUBN through bioinformatics analysis. The pathogenic effects of the potentially causative variants were verified utilizing complementary analysis of clinical data and systematic characterization of the variants' expression and function with clinical samples and in vitro experiments in HEK293T cell lines along with in vivo experiments in mice.Results: In this study, we identified four novel variants locating after the vitamin B12 (vitB12)-binding domain of Cubilin (encoded by CUBN, NM_001081.3: c.4397G > A (p.C1466Y), c.6796C > T (p.R2266X), c.6821 + 3A > G and c.5153_5154delCT (p.S1718X)) in two families. Moreover, the variants severely affected the expression and function of Cubilin in renal proximal tubules and caused albuminuria, increasing levels in urine transferrin and α1-microglobulin, but without progressive glomerular filtration barrier (GFB) impairment, vitB12 deficiencies or abnormal blood levels of HDL and albumin. Further mechanistic insights showed that the variants after the vitB12-binding domain of CUBN merely disrupted the association with Amnionless (AMN) that exhibited aberrant localization in cell cytoplasm rather than membrane.Conclusions: Here, our findings suggested that different mutation types after the vitB12-binding domain of CUBN uncouple proteinuria from glomerular filtration barrier, that may be an unexpectedly common benign condition in humans and may not require any proteinuria-lowering treatment or renal biopsy. [ABSTRACT FROM AUTHOR]

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

The regulation of iron metabolism in biological systems centers on providing adequate iron for cellular function while limiting iron toxicity. Because mammals cannot excrete iron, mechanisms have evolved to control iron acquisition, storage, and distribution at both systemic and cellular levels. Hepcidin, the master regulator of iron homeostasis, controls iron flows into plasma through inhibition of the only known mammalian cellular iron exporter ferroportin. Hepcidin is feedback-regulated by iron status and strongly modulated by inflammation and erythropoietic demand. This review highlights recent advances that have changed our understanding of iron metabolism and its regulation.

The iron-regulatory hormone hepcidin is induced early in infection, causing iron sequestration in macrophages and decreased plasma iron; this is proposed to limit the replication of extracellular microbes, but could also promote infection with macrophage-tropic pathogens. The mechanisms by which hepcidin and hypoferremia modulate host defense, and the spectrum of microbes affected, are poorly understood. Using mouse models, we show that hepcidin was selectively protective against siderophilic extracellular pathogens (Yersinia enterocolitica O9) by controlling non-transferrin-bound iron (NTBI) rather than iron-transferrin concentration. NTBI promoted the rapid growth of siderophilic but not nonsiderophilic bacteria in mice with either genetic or iatrogenic iron overload and in human plasma. Hepcidin or iron loading did not affect other key components of innate immunity, did not indiscriminately promote intracellular infections (Mycobacterium tuberculosis), and had no effect on extracellular nonsiderophilic Y enterocolitica O8 or Staphylococcus aureus Hepcidin analogs may be useful for treatment of siderophilic infections.

Purpose: mTOR regulates many normal physiological processes and when hyperactive can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling noninvasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need.Experimental Design: Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity.Results: After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that 89Zr-labeled transferrin (Tf) can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with PET. Finally, we show that 89Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard-of-care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer.Conclusions: In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues, and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man. Clin Cancer Res; 23(12); 3045-52. ©2016 AACR.

Green tea has been found to increase the lifespan of various experimental animal models including the fruit fly, Drosophila melanogaster. High in polyphenolic content, green tea has been shown to reduce oxidative stress in part by its ability to bind free iron, a micronutrient that is both essential for and toxic to all living organisms. Due to green tea's iron-binding properties, we questioned whether green tea acts to increase the lifespan of the fruit fly by modulating iron regulators, specifically, mitoferrin, a mitochondrial iron transporter, and transferrin, found in the hemolymph of flies. Publicly available hypomorph mutants for these iron regulators were utilized to investigate the effect of green tea on lifespan and fertility. We identified that green tea could not increase the lifespan of mitoferrin mutants but did rescue the reduced male fertility phenotype. The effect of green tea on transferrin mutant lifespan and fertility were comparable to w1118 flies, as observed in our previous studies, in which green tea increased male fly lifespan and reduced male fertility. Expression levels in both w1118 flies and mutant flies, supplemented with green tea, showed an upregulation of mitoferrin but not transferrin. Total body and mitochondrial iron levels were significantly reduced by green tea supplementation in w1118 and mitoferrin mutants but not transferrin mutant flies. Our results demonstrate that green tea may act to increase the lifespan of Drosophila in part by the regulation of mitoferrin and reduction of mitochondrial iron.

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe-/-) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway.

Trophic factor treatment has been shown to improve the recovery of brain and spinal cord injury (SCI). In this study, we examined the effects of TSC1 (a combination of insulin-like growth factor 1 and transferrin) 4 and 8 h after SCI at the thoracic segment level (T12) in nestin-GFP transgenic mice. TSC1 treatment for 4 and 8 h increased the number of nestin-expressing cells around the lesion site and prevented Wallerian degeneration. Treatment with TSC1 for 4 h significantly increased heat shock protein (HSP)-32 and HSP-70 expression 1 and 2 mm from lesion site (both, caudal and rostral). Conversely, the number of HSP-32 positive cells decreased after an 8-h TSC1 treatment, although it was still higher than in both, non-treated SCI and intact spinal cord animals. Furthermore, TSC1 increased NG2 expressing cell numbers and preserved most axons intact, facilitating remyelination and repair. These results support our hypothesis that TSC1 is an effective treatment for cell and tissue neuroprotection after SCI. An early intervention is crucial to prevent secondary damage of the injured SC and, in particular, to prevent Wallerian degeneration.

Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested that murine neonatal host defense against infection could be compromised by immunosuppressive CD71(+) erythroid splenocytes. We examined the impact of CD71(+) erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71(+) erythroid (CD235a(+)) cells in human neonates. Adoptive transfer or an Ab-mediated reduction in neonatal CD71(+) erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b(+) cells was not limited to neonatal splenocytes; it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 Ab showed reduced splenic bacterial load following bacterial challenge compared with isotype-treated mice. However, adoptive transfer of enriched CD71(+) erythroid splenocytes to CD71(+)-reduced animals did not reduce bacterial clearance. Human CD71(+)CD235a(+) cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71(+) erythroid splenocytes under these experimental conditions suggests that the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 Ab treatment, rather than a reduction in immunosuppressive CD71(+) erythroid splenocytes, was likely responsible for the reported enhanced bacterial clearance. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests that they may have a limited role in reducing inflammation secondary to microbial colonization.

Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.

Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a cost-effective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are ∼1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn't need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.

UnlabelledA noninvasive technology that indiscriminately detects tumor tissue in the brain could substantially enhance the management of primary or metastatic brain tumors. Although the documented molecular heterogeneity of diseases that initiate or eventually deposit in the brain may preclude identifying a single smoking-gun molecular biomarker, many classes of brain tumors are generally avid for transferrin. Therefore, we reasoned that applying a radiolabeled derivative of transferrin ((89)Zr-labeled transferrin) may be an effective strategy to more thoroughly identify tumor tissue in the brain, regardless of the tumor's genetic background.MethodsTransferrin was radiolabeled with (89)Zr, and its properties with respect to human models of glioblastoma multiforme were studied in vivo.ResultsIn this report, we show proof of concept that (89)Zr-labeled transferrin ((89)Zr-transferrin) localizes to genetically diverse models of glioblastoma multiforme in vivo. Moreover, we demonstrate that (89)Zr-transferrin can detect an orthotopic lesion with exceptional contrast. Finally, the tumor-to-brain contrast conferred by (89)Zr-transferrin vastly exceeded that observed with (18)F-FDG, currently the most widely used radiotracer to assess tumor burden in the brain.ConclusionThe results from this study suggest that (89)Zr-transferrin could be a broadly applicable tool for identifying and monitoring tumors in the brain, with realistic potential for near-term clinical translation.

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based transport to deliver essential cargo into dendrites. To test different models of differential motor protein regulation and to understand how different compartments in neurons are supplied with necessary functional proteins, we studied mechanisms of dendritic transport, using Drosophila as a model system. Our data suggest that dendritic targeting systems in Drosophila and mammals are evolutionarily conserved, since mammalian cargoes are moved into appropriate domains in Drosophila. In a genetic screen for mutants that mislocalize the dendritic marker human transferrin receptor (hTfR), we found that kinesin heavy chain (KHC) may function as a dendritic motor. Our analysis of dendritic and axonal phenotypes of KHC loss-of-function clones revealed a role for KHC in maintaining polarity of neurons, as well as ensuring proper axonal outgrowth. In addition we identified adenomatous polyposis coli 1 (APC1) as an interaction partner of KHC in controlling directed transport and modulating kinesin function in neurons.

The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin N-terminal homology (ENTH) domain has potent vesicle tubulation activity despite a lack of intrinsic molecular curvature. EPR revealed that the N-terminal alpha-helix penetrates the phosphatidylinositol 4,5-bisphosphate-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin 1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A-gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.

Aluminum is suspected to play a role in several neurological disorders. Reactive oxygen species (ROS) lead to oxidative stress, which is thought to be a possible mechanism for neurological damage. Interactions between aluminum and iron, a known promoter of prooxidant events, were studied in cerebral tissues using a fluorescent probe to measure rates of generation of ROS. Al2(SO4)3 alone failed to stimulate ROS production over a wide range of concentrations (50-1000 microM). The aluminum-deferrioxamine chelate in the absence of iron could also not potentiate ROS formation. However, Al2(SO4)3 potentiated FeSO4-induced ROS, with a maximal effect at 10 microM Fe and 500 microM Al. Kaolin, a hydrated aluminum silicate, did not potentiate iron-induced ROS formation. Ferritin had a minor stimulatory effect on ROS generation, but this was not potentiated by the concurrent presence of Al2(SO4)3. Transferrin had no effect on basal rates of ROS generation, but when Al2(SO4)3 was also present, ROS production was enhanced. It is concluded that: 1. There is a potentiation of iron-induced ROS by aluminum salts; 2. Free or complexed aluminum alone is not a key producer of ROS; and 3. High rates of ROS production are unlikely to be owing to the displacement by aluminum iron from its biologically sequestered locations.

Aluminum is suspected to play a role in several neurological disorders. Reactive oxygen species (ROS) lead to oxidative stress, which is thought to be a possible mechanism for neurological damage. Interactions between aluminum and iron, a known promoter of prooxidant events, were studied in cerebral tissues using a fluorescent probe to measure rates of generation of ROS. Al2(SO4)3 alone failed to stimulate ROS production over a wide range of concentrations (50-1000 microM). The aluminum-deferrioxamine chelate in the absence of iron could also not potentiate ROS formation. However, Al2(SO4)3 potentiated FeSO4-induced ROS, with a maximal effect at 10 microM Fe and 500 microM Al. Kaolin, a hydrated aluminum silicate, did not potentiate iron-induced ROS formation. Ferritin had a minor stimulatory effect on ROS generation, but this was not potentiated by the concurrent presence of Al2(SO4)3. Transferrin had no effect on basal rates of ROS generation, but when Al2(SO4)3 was also present, ROS production was enhanced. It is concluded that: 1. There is a potentiation of iron-induced ROS by aluminum salts; 2. Free or complexed aluminum alone is not a key producer of ROS; and 3. High rates of ROS production are unlikely to be owing to the displacement by aluminum iron from its biologically sequestered locations.

The extracellular phospholipase A2s (PLA2) from cobra venom, rattlesnake venom, and porcine pancreas were analyzed by radiation inactivation to determine their functional aggregation states. The analysis was performed in the presence of the protein transferrin at two different concentrations of PLA2: 5 micrograms/ml. The small size of these proteins necessitated the use of high radiation dosages. The catalytic activity of all samples decreased as a single exponential as a function of radiation dosage, to > 97% inactivation. Target size analysis of these curves yielded sizes corresponding to dimers for all three PLA2s, indicating that all three enzymes exist as dimers or larger aggregates under the conditions studied. An analysis of the amount of intact protein remaining by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the loss of protein also followed a dimeric size for all three PLA2s. The loss of protein as a dimer indicates that transfer of radiation energy is occurring between polypeptides.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.

It is not yet known what properties distinguish macrophages which can kill facultative intracellular bacteria, such as Listeria monocytogenes, from those which cannot. Listeria is an organism which requires iron for growth, yet macrophage listericidal mechanisms are also likely to be iron dependent. We show here that resident peritoneal macrophages and thioglycollate-elicited macrophages cannot kill listeria, but proteose peptone-elicited and FCS-elicited macrophages can. All these cell populations phagocytose listeria. Transferrin receptor expression is low on resident cells, intermediate on peptone- and FCS-elicited cells, and high on thioglycollate-elicited cells. Transferrin transports iron into cells via the transferrin receptor: thus, iron content of resident cells is low, of peptone- and FCS-elicited cells is intermediate, and of thioglycollate-elicited cells is high. Moreover, antibody to transferrin, which prevents it binding its receptor, inhibits listericidal macrophages from killing this bacterium. Finally, nonlistericidal cells with high transferrin receptor expression and high intracellular iron become listericidal if they are incubated with apotransferrin, an iron-free ligand which prevents iron uptake by cells. These data suggest that macrophages must have enough available intracellular iron to support listericidal mechanisms, but too much iron favors growth of the bacterium, which no longer can be killed by the macrophage.